Abcam’s MMP9 Human ELISA (Enzyme-Linked Immunosorbent Assay) kit is an in vitro enzyme-linked immunosorbent assay for the quantitative measurement of Human MMP9 pro and active forms in serum, plasma (Collect plasma using heparin as an anticoagulant. EDTA and Citrate are not recommended), and cell culture supernatants.

This assay employs an antibody specific for Human MMP9 coated on a 96- well plate. Standards and samples are pipetted into the wells and MMP9 present in a sample is bound to the wells by the immobilized antibody. The wells are washed and biotinylated anti-Human MMP9 antibody is added. After washing away unbound biotinylated antibody, HRP-conjugated streptavidin is pipetted to the wells. The wells are again washed, a TMB substrate solution is added to the wells and color develops in proportion to the amount of MMP9 bound. The Stop Solution changes the color from blue to yellow, and the intensity of the color is measured at 450 nm.

May play an essential role in local proteolysis of the extracellular matrix and in leukocyte migration. Could play a role in bone osteoclastic resorption. Cleaves KiSS1 at a Gly--Leu bond. Cleaves type IV and type V collagen into large C-terminal three quarter fragments and shorter N-terminal one quarter fragments. Degrades fibronectin but not laminin or Pz-peptide.

The conserved cysteine present in the cysteine-switch motif binds the catalytic zinc ion, thus inhibiting the enzyme. The dissociation of the cysteine from the zinc ion upon the activation-peptide release activates the enzyme.

Post-translationalmodifications

Processing of the precursor yields different active forms of 64, 67 and 82 kDa. Sequentially processing by MMP3 yields the 82 kDa matrix metalloproteinase-9.N- and O-glycosylated.

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75000 cells/well were seeded in a 6-well plate for 96 hours in serum-free medium with an induction with TNF-alpha alone or a mix of TNF-alpha and TGF-beta. Medium were collected and centrifuged at 2000 rpm for 10 minutes, then supernatants were transferred to 1.5 ml tubes and kept at -80°C until use.The concentrations of MMP9 were quantified with commercially available ELISA kit (Abcam, catalog number ab100610) according to the manufacturer´s protocol without any dilution.All samples were assayed in triplicates using a microplate reader and values were reported as ng/mL. The mean absorbance for each set of duplicate standards and samples was calculated. The standard curve was plotted in a log10-log10 scale.

Uterine aspirates were collected by aspiration with a Cornier Pipelle (Eurogine Ref. 03040200) in the office of the clinician or in the operating room prior to surgery and transferred to 1.5 ml microtubes. Phosphate buffer saline was added in a 1:1 (v/v) ratio and centrifuged at 2,500 rcf for 20 min in order to separate the soluble fraction (supernatant) from the solid fraction (pellet). The supernatants were kept at -80°C until use.The concentrations of MMP9 in the soluble fraction of uterine aspirates were quantified with commercially available ELISA kit (Abcam, catalog number ab100610) according to the manufacturer´s protocol . As no results are available regarding the levels of these proteins in uterine aspirates, samples were diluted using five serial dilutions: no dilution, 1:10, 1:100, 1:1000, 1:10000. The same amount of total protein from 6 uterine aspirate samples was loaded in each well. All samples were assayed in duplicates using a microplate reader and values were reported as ng/mL. The mean absorbance for each set of duplicate standards and samples was calculated. The standard curve was plotted in a log10-log10 scale (see figure 1A). The CV (%) between the duplicates of the samples ranged from 0-13% (average of 4%). Concentration of MMP9 of uterine aspirates ranged from 1 to 664 ng/u (see figure 1B).

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Submitted Apr 19 2016

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